Optogenetics is transforming how scientists study neural activation. Julie Manoharan shines a light on several research groups using optogenetics to better understand the neurophysiology of drug addiction.

Over 5 million Americans aged 12 and up abused cocaine in some form during
2008, making it the second most abused drug after marijuana.

A powerful stimulant of the central nervous system that reaches the brain
extremely quickly, cocaine binds to dopamine transporters, inhibiting their
usual and reuptake functions. When dopamine accumulates, it increases
dopiminergic receptor signaling and thereby produces a pleasurable
sensation. It also produces a desire for more.

Although the molecular mechanisms of addiction to drugs such as cocaine are
becoming better understood, what remains unclear is the specific neural
circuits in the brain that play a role. But a new technique called
optogenetics has emerged that might give new insights into the both the
molecular biology and neurophysiology of drug addiction.

The Deisseroth lab: “Let there be light”

In 2005, Stanford University researcher Karl Deisseroth could see the limits
of investigating psychiatric diseases with modern molecular biology tools.
Suspecting that these tools would never provide a deep-enough insight into
disease physiology, Deisseroth set out to develop a new technique to
implement temporal and spatial control over neuronal activation in free
moving animals using light. He called the technique “optogenetics.”

In an experiment by Karl Deisseroth at Stanford University, a mouse expressing light-activated proteins in its motor cortex starts running when the area is activated by blue light. Source: YouTube

To use optogenetic tools, researchers inject a virus into the brain to express light-activated proteins in specific neurons. An optical fiber is then inserted to activate those specific circuits. Above, circuits associated with Parkinson's disease are targeted. Source: NSF

Light-activated proteins are at the heart of optogenetics because they enable
the direct stimulation of neurons through the simple flashing of light. In
microorganisms such as algae and bacteria, these proteins allow cells to
mediate electrical charges on their membranes in response to visible light.
Deisseroth and his colleagues took advantage of this idea, reasoning that if
these proteins could be modified and then expressed in specific neurons,
then it might be possible to use light pulses to control the neural
circuitry of the brain, too.

“It was a very risky endeavor,” recalls Viviana Gradinaru, who was an eager
graduate student in Deisseroth’s lab during the development of the first
optogenetic toolkit. To modify the light-activated proteins for expression
in mammalian brains, it required a significant amount of capital without any
guarantee of success. “Most likely this shouldn’t have worked, but the
experiment was extremely successful. It was very exciting and impressive,”
she says.

To deliver these modified proteins, the researchers inject a virus containing
the coding sequence of the light-activated channel into the brain of the
subject—usually a mouse or rat. The virus is designed to target specific
neurons, and once infected with the modified light-activated protein code,
these neurons begin expressing the light-sensitive protein.

An optical fiber is then implanted into the target brain area to provide the
necessary light stimulation. Once the optical fiber in implanted, neurons
can be stimulated with blue light or inhibited with yellow light depending
on the light-activated channel transfected into the neuron. Most impressive
might be that the light can target neurons on the millisecond timescale, a
level of precision previously not available to neurobiologists.

What has been particularly exciting about the technique, according to
Gradinaru, is how easy it is. As a graduate student, she completed her first
optogenetics experiment after only one week of training, and Gradinaru says
that the ease of use gave their approach a competitive edge over others
working in optogenetics. Since finding its way into the spotlight,
Deisseroth has collaborated with many researchers from around the world,
teaching hundreds how to use the tool. The lab has published 37 papers
outlining potential uses of optogenetic tools for research on sleep
behavior, Parkinson’s disease, motor control, and escape behavior. But most
recently, optogenetics has been lighting up drug addiction studies, giving
researchers the means to look at specific circuits in the brain involved
with reward learning.

Shining the spotlight on dopamine

Antoine Adamantidis, a member of Stanford University’s Psychiatry and
Behavioral Sciences Department, is one of Deisseroth’s many collaborators.
Interested in further characterizing the role of dopaminergic neurons in
reward and addiction, he wanted to know if activation of these neurons alone
caused drug abuse behavior.

But as Deisseroth had found in his own research, traditional research tools
did not have the temporal and spatial precision to answer that question. For
example, electrical stimulation activates both dopaminergic neurons and the
surrounding neurons in the brain, making it unclear what exactly is
contributing to the behavior. His research often came up on dead-ends or
too-general results, and it wasn’t until optogenetics that he found the tool
to help him answer his question.

“You can really manipulate, as you want, the activity of neurons. You can
activate them, inhibit them, with very high precision,” says Adamantidis.

Using optogenetics, Adamantidis was able to control the stimulation of the
dopaminergic neurons of mice while they explored two chambers: one white,
the other black. While mice were in the white chamber, his research team
stimulated the mice’s dopaminergic neurons with 1 Hz of blue light. While
mice were in the black box, they used 50 Hz. In the end, the mice showed a
preference for the black box, suggesting that dopamine alone is enough to
induce reward behavior.

“It is a really promising technique,” says Adamantidis. “It’s also really fun
to play with in the lab, just because it’s so impressive.”

Illuminating dopamine receptor subtypes

Five types of dopamine receptors—D1, D2, D3, D4, and D5—are involved in
various neurological processes. Previous research has suggested that
subtypes D1 and D2 have opposite roles in motor behavior, but their role in
drug addiction has not been well characterized.

Stanford’s Antoine Adamantidis found that after optogenetic dopamine circuit stimulation during a visit to one chamber, mice preferred that chamber over two others wherein the circuit received either less stimulation or no stimulation at all.

This was a knowledge gap that Mary Kay Lobo, postdoctoral fellow at Mount
Sinai Medical Center, decided to fill. “We wanted to see what happens when
you actually activate [D1 and D2] during these behaviors, because there’s
just no information on that,” says Lobo.

The problem was that the D1 and D2 cells are located within the nucleus
accumbens (NAc), an interior region of the brain to which researchers lacked
adequate access in order to study the specific roles of D1 and D2 cell
subtypes. “The problem in the past has been that the cells are
heterogeneously intermixed in the brain region and it’s been hard to
separate them,” says Lobo. “Now we have these nice tools to separate them.”

These “nice tools” are the optogenetic tools that allowed Lobo and her
colleagues to activate D1 and D2 cell subtypes separately in mice while they
experienced a cocaine response. In her experiment, Lobo presented mice with
two chambers and conditioned them to prefer one over the other by rewarding
them with cocaine in one chamber. Then they introduced their optogenetic
tools. With the mice conditioned to prefer the cocaine chamber over the
other chamber, Lobo’s group activated the D1 receptors. The mice returned to
the cocaine chamber more frequently, increasing their level of addiction.
When the researchers activated the D2 receptors, the mice spent less time in
the cocaine chamber. That means that D1 and D2 dopamine receptor subtypes
play opposite roles in the expression of reward in cocaine response. D1
receptors activate reward behavior, while D2 receptors inhibit reward
behavior.

“The findings suggest that future therapies aimed at changing the activity of
the D2 neurons, making them more active, might help to decrease a
cocaine-addictive phenotype,” said Lobo.

Radiating relapse

Luis de Lecea, a professor of psychiatry and behavioral science at Stanford
University and another frequent collaborator of Deisseroth’s, has worked
extensively with optogenetics in numerous fields including sleep behavior
and drug addiction. Recently, he has applied the technique to better
understand relapse behavior.

“Optogenetics allows us to define the circuits much more precisely than before
and that can lead to better treatment for drug abusers,” Lecea told BioTechniques.

To test his theory that hypocretins—a pair of excitatory neuropeptide
hormones—are involved in the pleasurable response delivered by the brain
during relapse, Luis designed an experiment wherein previously addicted mice
had the choice of two levers. One lever optogenetically stimulated the
hypocretin circuit; the other did nothing. The mice preferred the lever that
activated the hypocretin circuit, and their drug seeking behavior returned,
suggesting hypocretin contributes to relapse behavior.

“We are really using optogenetics at its best,” Lecea said. “The cell
specificity and fine temporal resolution are really unprecedented in vivo.”
Using their findings, Lecea believes that one day a treatment may be
developed to inhibit the activity of hypocretin in humans, which would in
turn inhibit the cravings in those addicted and reducing the risk of
relapse.

A burgeoning technique already showing promise across a variety of
disciplines, optogenetics is primed to answer some exciting questions about
neural circuits and behavior. “It can really answer some big questions in
the field, and it can open our eyes to new perspective and a new way of
testing the hypotheses we have,” Adamintidis reflected. “The funny thing
about optogenetics is that we have to rethink the whole way we were
considering neuronal behavior. We can now read behavior on a millisecond
timescale. Our tools are just so much better.”

---

2010 marked the beginning of our Methods-specific Newsletter series. Covering
cell culture, microscopy, PCR, and antibody technology, BioTechniques
brought you the latest methodological and technical advances in these
exciting fields through weekly feature articles and news stories. If you
enjoyed the Top Microscopy Feature of 2010, check out the rest of the
editors’ picks of our favorite methods-specific news features from 2010 here.